Co-culturing BC Cells: Culturing Breast Cancer Cells with Bone Fragments to Study Cancer Cell Proliferation
Transkript
Start by placing a piece of bone wax into two cell culture plates labeled as test and control. Pipette an appropriate amount of bioluminescence expressing breast cancer cells at each plate center and incubate the plates for a specific duration to aid cell attachment. Place a small bone explant on the bone wax in the test plate and secure it by applying pressure.
Slowly pour a cell culture medium with fetal bovine serum, a growth supplement, to each plate side to submerge the explant and incubate at 37 degrees Celsius for a specific duration. The bone tissue in the test plate releases soluble factors, such as cytokines and SRC kinase, which attract breast cancer cells, resulting in their migration. Add a substrate to each plate and place the plates on a bioluminescence imaging chamber.
The cancer cell expressing bioluminescent protein oxidizes the substrate and emits light. Quantify proliferation by measuring total photons emitted by each cell using the software. Cancer cells in the test plate should proliferate faster than the cells in the control plate. In the following protocol, we will co-culture breast cancer cells adjacent to femur bone explants to measure breast cell proliferation.
For this experiment, prepare a breast cancer cell suspension with 100,000 cells per 50 microliters of DMEM plus 10% FBS. In addition, prepare plugs of bone wax for immobilizing the bone fragments. Using the cut off ends of a micropipette tip, store the plugs in a petri dish. Then with forceps transfer the plugs into a six-well plate and using a sterile glove, press the plugs into the 12 o'clock position.
Next, pipette 50 microliters of the cell suspension into the center of each well. Place the plate in a tissue culture incubator for 45 minutes to promote cell attachment. While the plate incubates, extract the bone fragments. With the cells attached to the plate, place the bone tissue fragments onto the bone wax in 3 wells, and press each down using the rongeur. Three wells without fragment serve as controls.
Next, slowly add five milliliters of DMEM plus 10% FBS to the wall of each well. The bone wax and bone fragments mustn't be dislodged. Then incubate the culture for 20 to 24 hours. The next day, image the cells. First add 300 micrograms per milliliter of luciferin to each well, and then immediately image the plate with an IVIS imaging Platform.
